Biology subjects

Kazmierczak, B. I.

Publications and source records attributed to Kazmierczak, B. I..

6 recordsLinked to original sources

bioRxiv2025

Injectisome assembly primes Pseudomonas aeruginosa for Type III secretion

Many Gram-negative pathogens, including Pseudomonas aeruginosa, use a Type III Secretion System (T3SS) to intoxicate eukaryotic cells. The T3SS is an important virulence factor linked to increased morbidity and mortality in infections, yet its expression slows bacterial growth and activates innate immune receptors. T3SS genes are expressed heterogeneously, with T3SS-ON cells arising from primed bacteria that express the T3SS transcriptional activator ExsA and respond immediately to T3SS activating signals. However, the mechanistic basis for priming is not known. ExsA is part of a complex protein-sequestration network, positively regulating its own expression as well as that of its anti-activator (ExsD), its anti-anti-activator (ExsC) and ExsCs binding partner (ExsE). These four proteins create a bistable regulatory network. We hypothesized that transcription from a cAMP-dependent promoter upstream of ExsA could drive cells into the primed state and tested this at the single cell level. Exogenous cAMP increased the proportion of primed, ExsA-expressing cells, with whole cell cryo-electron tomography demonstrating assembly of T3SS injectisomes under these conditions. Intrastrain variation in endogenous cAMP levels correlated with strain-specific proportions of primed bacteria, while genetic manipulation of adenylate cyclases and cAMP phosphodiesterase altered primed population size. Priming occurred in planktonically cultured populations and was independent of type IV pilus assembly or retraction; however, mutation of genes in the Pil-Chp system, which regulates cAMP production, changed proportions of primed cells. This work demonstrates how endogenous and exogenous cAMP inputs into a bistable regulatory switch generate subpopulations of T3SS-primed cells poised to respond to activating signals. ImportanceType III Secretion Systems (T3SS) are specialized protein secretion systems that allow bacteria to inject toxins into eukaryotic cells. T3SS are important virulence factors, but their expression carries a fitness cost: they slow bacterial growth and make bacteria vulnerable to detection by the innate immune system. Some pathogens, like Pseudomonas aeruginosa, balance the costs and benefits of T3SS expression by restricting T3SS expression to a subset of cells. T3SS-ON cells arise from primed bacteria that express the transcriptional activator ExsA and respond immediately to T3SS activating signals. However, the mechanistic basis for priming is unknown. In this study we tested whether expression of ExsA from a cAMP-dependent promoter could drive cells into the primed state and found this to be true. Whole cell cryo-electron tomography demonstrated that bacteria assembled T3SS injectisomes under these conditions. This work demonstrates how cAMP inputs into a bistable regulatory switch generate subpopulations of T3SS-primed cells.

microbiology↗

bioRxiv2025

A high-throughput, image-based assay to assess drug sensitivity of Acanthamoeba castellanii cysts.

Pathogenic free-living amoebae (FLA) such as Acanthamoeba castellanii are present in soil and water worldwide. A. castellanii causes systemic infections with very high mortality rates, yet drugs specifically targeting this pathogen are not available. Methods to reliably generate and assay cysts, which drive infection recurrence and drug resistance, are unavailable in a high-throughput format suitable for drug screening and testing. In this study, we developed a robust and reproducible protocol for encysting A. castellanii as well as a high-throughput, quantitative cyst viability assay using fluorescent live/dead staining coupled with microscopy and automated image analysis. These methods were used to screen the cysticidal activity of 17 clinically relevant drugs and disinfectants and identified five agents, including caspofungin, as active against cysts.

microbiology↗

bioRxiv2025

An improved viability assay for Acanthamoeba castellanii trophozoites reveals drug-induced pseudocyst formation

Acanthamoeba castellanii is a free-living amoeba (FLA) that causes fatal human infections with few effective treatments. One limitation of new drug development is the lack of accurate, high-throughput, and quantitative viability assays that score both trophozoites and cysts. A colorimetric assay using Sulforhodamine B (SRB), which measures cell adherence, has previously been adapted for A. castellanii trophozoites. In this study, we demonstrated that the SRB assay can be optimized to serve as a robust, high-throughput platform that can measure viable Acanthamoeba trophozoites, pseudocysts and cysts. We used this assay to measure the IC50 of 18 commonly used drugs and disinfectants on A. castellanii trophozoites, demonstrating that several clinically used drugs induce pseudocyst formation rather than amoeba death.

microbiology↗

bioRxiv2024

Non-genetic adaptation by collective migration

Cell populations must adjust their phenotypic composition to adapt to changing environments. One adaptation strategy is to maintain distinct phenotypic subsets within the population and to modulate their relative abundances via gene regulation. Another strategy involves genetic mutations, which can be augmented by stress-response pathways. Here, we studied how a migrating bacterial population regulates its phenotypic distribution to traverse diverse environments. We generated isogenic Escherichia coli populations with varying distributions of swimming behaviors and observed their phenotype distributions during migration in liquid and porous environments. We found that the migrating populations became enriched with high-performing swimming phenotypes in each environment, allowing the populations to adapt without requiring mutations or gene regulation. This adaptation is dynamic and rapid, reversing in a few doubling times when migration ceases. By measuring the chemoreceptor abundance distributions during migration towards different attractants, we demonstrated that adaptation acts on multiple chemotaxis-related traits simultaneously. These measurements are consistent with a general mechanism in which adaptation results from a balance between cell growth generating diversity and collective migration eliminating under-performing phenotypes. Thus, collective migration enables cell populations with continuous, multi-dimensional phenotypes to flexibly and rapidly adapt their phenotypic composition to diverse environmental conditions. Significance statementConventional cell adaptation mechanisms, like gene regulation and stochastic phenotypic switching, act swiftly but are limited to a few traits, while mutation-driven adaptations unfold slowly. By quantifying phenotypic diversity during bacterial collective migration, we discovered an adaptation mechanism that rapidly and reversibly adjusts multiple traits simultaneously. By balancing the generation of diversity through growth with the loss of phenotypes unable to keep up, this process tunes the phenotypic composition of migrating populations to the environments they traverse, without gene regulation or mutations. Given the prevalence of collective migration in microbes, cancers, and embryonic development, non-genetic adaptation through collective migration may be a universal mechanism for populations to navigate diverse environments, offering insights into broader applications across various fields.

systems biology↗

bioRxiv2023

Identification of efflux substrates using a riboswitch-based reporter in Pseudomonas aeruginosa

Pseudomonas aeruginosa is intrinsically resistant to many classes of antibiotics, reflecting the restrictive nature of its outer membrane and the action of its numerous efflux systems. However, the dynamics of compound uptake, retention and efflux in this bacterium remain incompletely understood. Here, we exploited the sensor capabilities of a Z-nucleotide sensing riboswitch to create an experimental system able to identify physicochemical and structural properties of compounds that permeate the bacterial cell, avoid efflux, and perturb the folate cycle or de novo purine synthesis. In a first step, a collection of structurally diverse compounds enriched in antifolate drugs was screened for ZTP riboswitch reporter activity in efflux-deficient P. aeruginosa, allowing us to identify compounds that entered the cell and disrupted the folate pathway. These initial hits were then rescreened using isogenic efflux-proficient bacteria, allowing us to separate efflux substrates from efflux avoiders. We confirmed this categorization by measuring intracellular levels of select compounds in the efflux-deficient and - proficient strain using high resolution LC-MS. This simple yet powerful method, optimized for high throughput screening, enables the discovery of numerous permeable compounds that avoid efflux and paves the way for further refinement of the physicochemical and structural rules governing efflux in this multi-drug resistant Gram-negative pathogen. ImportanceTreatment of Pseudomonas aeruginosa infections has become increasingly challenging. The development of novel antibiotics against this multi-drug resistant bacterium is a priority, but many drug candidates never achieve effective concentrations in the bacterial cell due due to its highly restrictive outer membrane and the action of multiple efflux pumps. Here, we develop a robust and simple reporter system in P. aeruginosa to screen chemical libraries and identify compounds that either enter the cell and remain inside, or enter the cell and are exported by efflux systems. This approach enables developing rules of compound uptake and retention in P. aeruginosa that will lead to more rational design of novel antibiotics.

microbiology↗

bioRxiv2019

Chemical Impacts of the Microbiome Across Scales Reveal Novel Conjugated Bile Acids

A mosaic of cross-phyla chemical interactions occurs between all metazoans and their microbiomes. In humans, the gut harbors the heaviest microbial load, but many organs, particularly those with a mucosal surface, associate with highly adapted and evolved microbial consortia1. The microbial residents within these organ systems are increasingly well characterized, yielding a good understanding of human microbiome composition, but we have yet to elucidate the full chemical impact the microbiome exerts on an animal and the breadth of the chemical diversity it contributes2. A number of molecular families are known to be shaped by the microbiome including short-chain fatty acids, indoles, aromatic amino acid metabolites, complex polysaccharides, and host lipids; such as sphingolipids and bile acids3-11. These metabolites profoundly affect host physiology and are being explored for their roles in both health and disease. Considering the diversity of the human microbiome, numbering over 40,000 operational taxonomic units12, a plethora of molecular diversity remains to be discovered. Here, we use unique mass spectrometry informatics approaches and data mapping onto a murine 3D-model13-15 to provide an untargeted assessment of the chemical diversity between germ-free (GF) and colonized mice (specific-pathogen free, SPF), and report the finding of novel bile acids produced by the microbiome in both mice and humans that have evaded characterization despite 170 years of research on bile acid chemistry16.

microbiology↗